dNTP Imbalance and DNA Double Strand Breaks in Mouse FM3A Cells and the Mechanism of Cell Death

本文研究了脱氧核糖核苷三磷酸(DNTP)池失衡诱导培养的小鼠FM3A细胞死亡的机制。我们以前报道过5-氟代脱氧尿苷(FdUrd)可以引起细胞内dNTP池的失衡，继而导致DNA双链断裂和随后的细胞死亡。我们现在发现，FdUrd诱导的DNA片段可以分为两类。一种是大的DNA片段，大小为100-200kBP，相当于哺乳动物基因组中的复制单位；另一种是较短的DNA片段，其间距为核小体长度，是细胞凋亡的特征。此外，我们还从FdUrd处理的FM3A细胞的裂解物中纯化了导致双链断裂的内切酶。在FdUrd处理的FM3A细胞的裂解物中可检测到这种内切酶活性，但在未处理的细胞中检测不到这种内切酶活性。经纯化后，酶的相对分子质量约为40 kDa，最适pH为6.0~6.5，不需要二价金属阳离子。核酸内切酶对DNA的切割产生5‘-磷酸末端。具有这些性质的哺乳动物细胞的内切酶在文献中没有描述。我们怀疑这种内切酶(我们称之为S内切酶)在dNTP不平衡死亡中起着重要作用，这一过程在许多方面类似于细胞自杀反应--“细胞凋亡”。

The mechanism of deoxyribonucleoside triphosphate (dNTP) pool imbalanceinduced cell death in cultured mouse FM3A cells was studied. We reported previously that 5-fluorodeoxyuridine (FdUrd) can induce intracellular dNTP pool imbalance followed by DNA double-strand breaks and subsequent cell death. We have now found that the FdUrd-induced DNA fragments can be separated into two classes. One was large DNA fragments with sizes of 100-200 kbp, which corresponded to the replication units in mammalian genomes, and the other was shorter DNA fragments with a pitch of nucleosome length, characteristic for apoptosis. In addition, we have purified the double-strand break causing endonuclease from the lysate of FdUrd-treated FM3A cells. This endonuclease activity was detectable in the lysate of FdUrd-treated FM3A cells but not in untreated cells. The endonuclease was purified to near homogeneity and its molecular mass was estimated to be approximately 40 kDa by use of a DNA-containing SDS-PAGE.The endonuclease exhibited an optimal pH of 6.0-6.5 and did not require divalent metal cations for its activity. The cleavage of DNA by the endonuclease produced 5'-phosphoryl termini. Endonuclease of mammalian cells having these properties have not been described in the literature. We suspect that this endonuclease (which we termed Endonuclease S) plays an important role in the dNTP imbalance death, a process in many ways similar to 'apoptosis', the cellular suicide response.